The overall goal of this research is to understand the relationship between PBP structure, penicillin binding, and the resistance of S. aureus to penicillins. To meet this goal the investigator outlines the following three specific aims: [1] To clone, sequence, and map PBP genes. The investigator already has cloned the genes encoding PBPs 2 and 4. The gene encoding PBP 1 will be identified, possibly by use of putative sequence data for it recorded in GenBank and cloned using a strategy involving probe hybridization of a S. aureus genomic library or PCR of chromosomal DNA. The gene encoding PBP 3 will be identified and cloned by screening an expression library with antisera raised against PAGE-purified PBP3 in a manner analogous to what they already have done for PBP2. The genes will be sequenced and physically mapped to a specific site on the chromosome by determining which SmaI, CspI, and BamHI fragments hybridize by Southern blotting. PBP gene sequences from representative strains will be compared to identify mutations associated with structural modification, altered penicillin binding, and resistance. [2] To examine the contribution of altered PBPs to the resistance phenotype. Recombinant PBP genes with one or more mutations associated with resistance will be used to create isogeneic strains from a common S. aureus strain 8325 background by allelic replacement (with production of the altered PBP instead of the native one) if possible, or alternatively, by transformation (with production of both the native and altered PBP). The effect of differences in sequence upon the resistance phenotype will be determined in standard susceptibility tests. [3] To assay beta-lactam binding of recombinant PBPs. Mutant and wild-type PBP genes will be subcloned into an E. coli expression vector to obtain soluble recombinant protein for use in penicillin-binding assays. The kinetics of binding will be determined.